RNA probe for detecting c-fes mRNA

ABSTRACT

A recombinant plasmid and an RNA sequence expressed by said plasmid are described. The RNA sequence hybridize specifically with human c-fes mRNA.

[0001] The present invention is related generally to diagnostic tests.More particularly, the present invention is related to an RNA probe fordetecting the presence of c-fes mRNA in biological samples, such ashuman cell and tissue RNA preparations.

[0002] Expression of the c-fes oncogene is known to play a certainfunctional role in myelopoiesis in henatopoietic cells (Smithgall et al,1988, J. Biol. Chem. 263, 15050-40 15055; Greer et al, 1988, Mol. Cell.Biol., 8, 578-587). However, heretofore direct evidence was lacking toprove that the expression of human c-fes gene induced myeloiddifferentiation in cells. Furthermore, a specific and sensitive assay tomeasure the level of c-fes mRNA in human cells and tissues was alsoheretofore not available.

SUMMARY OF THE INVENTION

[0003] It is, therefore, an object of the present invention to provide akit for the detection of c-fes mRNA in biological samples such as humancell and tissue RNA preparations.

[0004] It is a further object of the present invention to provide an RNAprobe for detecting the presence of c-fes mRNA in vitro or in situ.

[0005] It is another object of the present invention to provide arecombinant plasmid comprising exon 2 of the human c-fes genomicsequence for the expression of the transcription product of the c-fesoncogene in a suitable expression vector.

[0006] Other objects and advantages will become evident from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] These and other objects, features and many of the attendantadvantages of the invention will be better understood upon a reading ofthe following detailed description when considered in connection withthe accompanying drawings wherein:

[0008]FIG. 1 shows the various elements of the human c-fes genomicclone.

[0009]FIG. 2 shows schematic construction of the recombinant plasmidpcfes4ZKB.

[0010]FIG. 3 schematically shows various steps involved in the RNaseprotection assay with the c-fes RNA probe in accordance with the presentinvention.

[0011]FIG. 4 shows non-denaturing gel assay for p93^(c-fes) tyrosinekinase activity in colonies of K562 cells stably transfected withpECE/fes. K562 cells were cotransfected with pECE/fes and pSV2/neo as aselectable marker and G418 -resistant colonies were selected andscreened for p93^(c-fes) tyrosine kinase activity. Aliquots of membraneproteins (15 μg) present in 1.0% Triton X-100 cell extracts were assayedfor tyrosine kinase activity using the non-denaturing gel assaydescribed in the text.

[0012]FIG. 5 shows the comparison of tyrosine kinase activity in colonyWS-1 with differentiated HL-60 cells. One percent Triton X-100 extractswere prepared from either wild type K562 cells (“K562”),pSV2/neo-transfected K562 cells (“K562/neo”), colony WS-1 (“K562/fes”),or HL-60 cells treated for 4 days with 1.6% Me₂SO, and p93^(c-fes)tyrosine kinase was partially purified by tyrosine-agarosechromatography. Eluates (3 μg of protein) were assayed for tyrosinekinase activity using the non-denaturing gel assay as described in thetext.

[0013]FIG. 6 shows the Southern blot analysis of colonies of K562 cellsstably transfected with pECE/fes. DNA (10 μg) was prepared from wildtype K562 cells (“K562”) and selected colonies of cells transfected withc-fes (designated as “WS-1, WS-5, WS-6, WD-1, WD-2, WD-3, WD-4, andWD-7”), and digested with Eco RI. Plasmid p80 DNA (“p80”) containing the13.2 kb c-fes gene served as a control. After electrophoresis in 1%agarose gels, Southern blots were prepared and hybridized with a v-fesprobe as described herein below. Levels of c-fes integration relative towild type K562 cells were determined by laser densitometry of the 13.2kb Eco RI fragment. The endogenous K562 c-fes gene is not visible in theexposure shown (12 h); determination of the c-fes gene in wild typecells required longer autoradiographic exposure (>48 h; data not shown).

[0014]FIG. 7 shows the Southern blot analysis of a restriction digest ofDNA prepared from colony WS-1. DNA (10 μg) was prepared from wild typeK562 cells (“K562”) and colony WS-1 (“K562/fes”) and digested with EcoRI and Xho I. Plasmid p80 DNA (“p80”) containing the 13.2 kb c-fes geneserved as a control. Southern blots and hybridization were carried outas described in the text.

[0015]FIG. 8 shows the RNase protection assay of parental and clonalvariants of K562 cells stably transfected with pECE/fes. Poly-A RNA wasselected from 250 μg of total RNA prepared from wild type HL-60 cells(“HL-60”), wild type K562 cells (“K562”), and colonies WS-1(“K562/WS-1”), WS-5 (“K562/WS-5”) and WS-6 (“K562/WS-6”). Solutionhybridization was carried out with 10° cpm of a ^(ox)P-labeled c-fesantisense RNA probe containing the 222 bp sequence complementary to exon2 of the human c-fes gene. Following overnight incubation, thehybridization reaction was digested with RNase and the protected dsRNAfragments were resolved by electrophoresis on 6% polyacrylamide-ureagels, and visualized by autoradiography.

[0016]FIG. 9 shows the immunoprecipitation of parental and clonalvariants of K562 cells stably transfected with pECE/fes. Cell extractswere prepared from wild-type K562 cells. transfected clones WS-1 andWS-5, and HL-60 cells labeled with [^(oo)S ]Methionine, and p93^(c-fes)was immunoprecipitated with an anti-v-fes monoclonal antibody.Immunoprecipitates were analyzed by SDS-polyacrylamide gelelectrophoresis and autoradiography as described in the text. Thecontrol lane shows precipitation of HL-60 extracts in the absence of themonoclonal antibody.

[0017]FIG. 10 shows the growth curve of K562/fes clone WS-1, K562/neoand parental K562 cells. Wild type K562 cells (“K562”),pSV2/neo-transfected cells ( “K562/neo” ) and colony WS-1 (“K562/fes”)were grown for one week, and cells number was determined at one dayintervals with a Coulter particle counter. Cell viability was greaterthan 95% as determined by trypan blue exclusion.

[0018]FIG. 11 shows the photomicrographs of parental K562 cells andK562/fes clone WS-1. Parental K562 cells (A,C,E) and c-fes-transfectedclone WS-1 (B,D,F) were tested for their response to 2 day treatmentwith 10⁻⁷ M TPA (A,B), for their ability to reduce NBT (C,D) or fortheir capacity to phagocytize sheep erythrocytes (E,F).

DETAILED DESCRIPTION OF THE INVENTION

[0019] The above and various other objects and advantages of the presentinvention are achieved by a specific RNA sequence which hybridizes onlywith c-fes mRNA. said specific RNA sequence being obtained from theexpression of the recombinant plasmid pcfes4ZKB in a suitable expressionvector, such as E. coli, yeast, viruses and other prokaryotic oreukaryotic vectors well known to one of ordinary skill in the art.

[0020] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedhereunder are incorporated herein by reference. Unless mentionedotherwise, the techniques employed herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

MATERIALS AND METHODS

[0021] Materials—All radioisotopes were obtained from Du Pont-NewEngland Nuclear, Boston, Mass. Tyrosine-agarose, Me₂SO, andpoly(glu,tyr)₄₁₂ were purchased from Sigma, St. Louis, Mo. The v-fesprobe (460 bp Pst I-Pst I fragment) was purchased from Oncor,Gaithersburg, Md. Rabbit antisera to a recombinant c-fes peptide wasprovided by Dr. Dennis J. Slamon, UCLA School of Medicine, Los Angeles,Calif. Geneticin (G418) was purchased from Gibco, Grand Island, N.Y.Plasmids p80 and pSV2/neo were obtained from the American Type CultureCollection, Rockville, Md. The Mac-1 monoclonal antibody against themacrophage-specific differentiation was obtained from Hybritech, SanDiego, Calif. The monoclonal antibody (Ab-1) directed against the festransforming protein common to both the Snyder-Theilen and Gardnerstrains of feline sarcoma virus was purchased from Oncogene Sciences,Manhasset, N.Y.

[0022] Cell Culture—HL-60, K562, and Cos-1 cells were obtained from theAmerican Type Culture Collection. HL-60 and K562 cells were grown inRPMI-1640 medium supplemented with 10% heat-inactivated fetal calfserum, 40 mM Hepes, PH 7.4, 1 mM sodium pyruvate, nonessential aminoacids, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cos-1 cellswere grown in Dulbecco's Modified Eagle's medium supplemented as above.All cells were subcultured twice weekly, and maintained at a density of10⁵-10⁶ cells/mi. HL-60 cells were treated with 1.6% Me₂SO for 4 days toinduce granulocytic differentiation.

[0023] Preparation of Cell Extracts—Cells (0.5-1.0×10⁸) were collectedby centrifugation and washed twice in Hank's balanced salt solutioncontaining 20 mM EDTA without Mg²⁻or Ca²⁻. The cell pellet was sonicatedfor 5 seconds in 0.5 ml of 50 mM Tris-HCl (pH 7.5) containing 2 mM EGTA,10 mM DTT, 0.1% Triton-X 100, 1 mM PMSF, 50 μg/ml aprotinin, 200 μg/mlleupeptin and 400 μg/ml soybean trypsin inhibitor, and centrifuged at15,000×g at 4° C. for 10 min. The supernatant was removed and the pelletwas re-extracted with an identical buffer containing 1% Triton X-100.Protein concentrations were determined using a Coomassie blue-basedreagent (Pierce Chemical Co.) and BSA as a standard.

[0024] Non-denaturing Gel Assay for Tyrosine Kinase Activity—Tyrosinekinase activity present in crude cell extracts and column fractions wasassayed by non-denaturing polyacrylamide gel electrophoresis asdescribed by Glazer et al (1987) Anal. Biochem. 164, 214-220. Briefly,protein samples were subjected to electrophoresis in 4.5% polyacrylamidemini-gels (Hoefer Scientific) at 4° C. Following electrophoresis, thegels were incubated with Mg²⁻, Mn²⁻and [t-³²P]ATP in the presence andabsence of poly(glu,tyr)_(4:1), a synthetic polymer substrate in whichtyrosine acts as sole phosphate acceptor. Following incubation at 37° C.for 30 min, the gels were washed extensively in 5% trichloroacetic acidcontaining 10 mM sodium pyrophosphate, dried and kinase activity wasquantitated by autoradiography.

[0025] Tyrosine-agarose Chromatography, Immunoblotting, andImmunoprecipitation—One percent Triton X-100 extracts were furtherfractionated by tyrosine-agarose chromatography (Yu et al, 1987, J.Biol. Chem. 262. 17543-17548). Extracts were applied to 1.5 mltyrosine-agarose columns and aliquots (10 μg of protein) present in theeluate were resolved on 7.5% SDS-polyacrylamide mini-gels using themLaemmli buffer system (Laemmli, U. K., 1970, Nature 227, 680-685).Proteins were transferred to nitrocellulose membranes using the Genieelectrophoretic blotter (Idea Scientific). Immunoreactive p93^(c-fes)was visualized using antiserum to a recombinant human c-fes peptide, andthe Protoblot detection system (Promega Biotec) as described by themanufacturer. For immunoprecipitation, 5 ×10⁷ cells were labeled byincubation at 37° C. for 18 h in 3 ml of methionine-free Iscove'sModified Dulbeccol's Medium containing 5% fetal calf serum and 200μCi/ml [³⁵S]-methionine (1,140 Ci/mmol). Cells were then washed, lysedand subjected to immunoprecipitation with biotinylated anti-v-fesmonoclonal antibody (Veronese et al, 1982, J. Virol. 43, 896-904) andstreptavidin-agarose according to the manufacturer's protocol. Followingextensive washing, immune complexes resolved by electrophoresis through8% SDS-polyacrylamide gels. Gels were treated with Fluoro-Hance(Research Products International, Mount Prospect, Ill.) prior toautoradiography at −80° C.

[0026] Construction of the expression vector pECE/fes—An SV40-basedmammalian expression vector pECE (Ellis et al, 1986, Cell 45, 721-732)was provided by Dr. William J. Rutter, University of California, SanFrancisco. pECE was digested with Eco RI and dephosphorylated with calfintestine alkaline phosphatase. The entire human c-fes genomic sequencewas isolated as a 13.2 kb Eco RI fragment from the plasmid vector p80(Trus et al, 1982, J. Biol. Chem. 257, 2730-2733) and cloned into theexpression vector PECE. The orientation of c-fes was determined bySouthern blots using the v-fes probe and it was found to be in thecorrect orientation such that transcription of the c-fes coding sequenceis directed from the SV40 early promoter. This recombinant plasmid isdesignated pECE/fes.

[0027] Transfection of Cos-1 and K562 cells—Cos-1 cells (5×10⁵ cells/100mm plate) were transfected with 20 μg of pECE/fes by the modifiedcalcium phosphate precipitation procedure described by Chen and Okayama(1987) Mol. Cell. Biol. 7, 2745-2752. For transient expression. analysiswas performed 48 h posttransfection. K562 cells were transfected by theprotoplast fusion technique (Yoakum, G. H., 1984, BioTechniques 2,24-31). Briefly, 100 ml of an overnight culture of E. coli transformedwith pECE/fes and pSV2/neo were centrifuged at 4000×g for 15 min. Thebacteria were incubated for 2 hr at room temperature (22°-24° C.) with 3ml of a freshly prepared lysozyme solution (10 mg/ml in 20 mM Hepes, 20%sucrose, pH 7.1). The incubation was stopped by adding 0.8 ml of 1.25 MCaCl₂ and the protoplast preparation was diluted to 10 ml withRPMI-1640. K562 cells (5×10⁶ cells) were collected by centrifugation andtreated for 1.5 min with 2 ml of the protoplast preparation and 1 ml offresh 48% polyethylene glycol (mol wt 1000). The cells were then washedfive times with RPMI-1640 medium in a CO₂ incubator with the mediumchanged daily for the first two days. After 48 hr, cells were split andplated at 10³ cells per 100 mm plate containing RPMI-1640 mediumsupplemented with 20% heat-inactivated fetal calf serum and 0.4% agarose(SeaPlaque, FMC) and 2.5 mg/ml G418 (Gibco) for selection. After about14 days of incubation, colonies were selected and cultured in RPMI-1640medium with 0.2 mg/ml G418.

[0028] Southern Blot—High molecular weight DNA, prepared by theGross-Bellard method (Gross-Bellard et al, 1972, Eur. J. Biochem. 36,32-39) was digested with either Eco RI or Xho I, separated in 0.8%agarose gels and transferred to nitrocellulose (Southern, E. M., 1975,J. Mol. Appl . Genet. 1, 327-341). Hybridization was carried out withthe v-fes probe labeled with [α−³²P]dCTP by the random primer procedure(BRL) at 36° C. for 16 hr in 50% formamide, 5X SSC, 0.5% SDS, 5XDenhardt's solution and 100 μg/ml denatured salmon sperm DNA. Blots werewashed with 0.1X SSC, 0.1SDS at 65° C.

[0029] Cloning of genomic c-fes fragments for riboprobe synthesis—A 461bp Kpn I-Bgl II fragment of the human c-fes locus (Roebroek et al, 1985,EMBO. J. 4, 2897-2903) containing exon 2 and some 3′ and 5′ intronsequences, was cloned into the polylinker region of pGEM-4Z (PromegaBiotec). This vector contains the bacteriophage T7 promoter immediatelydownstream from and in an opposite orientation to the cloning site,allowing for preparation of a c-fes riboprobe (antisense RNAtranscript). This was accomplished by linearization of the vector 5′ tothe c-fes insert and incubation with T7 RNA polymerase, [α−³²P]CTP andunlabeled nucleoside triphosphates according to the manufacturer'sprotocol. The resulting riboprobe is 498 nucleotides in length, as itcontains some sequences transcribed from the vector template.

[0030] Poly-A⁺ RNA Isolation and RNase Protection Assay—Total cellularRNA was prepared by guanidinium isothiocyanate extraction of 10 ⁵ cellsfollowed by centrifugation through cesium chloride (Chirgwin et al,1979, Biochemistry 18, 5294-5298; Glisin et al, 1973, Biochemistry 13,2633-2641). Poly-A⁻ RNA was selected from 250 μg total RNA by batchadsorption to oligo-dT cellulose (New England Biolabs). The fractioneluting from oligo-dT cellulose selection was hybridized with 10⁶ cpm ofthe ^(a2)P-labeled c-fes riboprobe (see above). Following overnight(about 12-16 hrs) incubation, the hybridization reaction was digestedwith RNase, and the protected dsRNA fragments were resolved bypolyacrylamide-urea gel electrophoresis and visualized byautoradiography.

[0031] Histochemical Assays—Lysozyme activity was measuredspectrophotometrically at 450 nm by the lysis of M.lysodeikticus(Selsted et al, 1978, Infection and Immunity 20, 782-791).The ability of cells to reduce NBT to formazan was assessed by themethod described by Breitman et al (1980) Proc. Natl. Acad. Sci. U.S.A.77, 2936-2940. Fc receptors and immunophagocytosis were determined usingsheep erythrocytes coated with anti-erythrocyte antibodies (Breitman etal, 1984, in Methods for Serum-Free Culture of Neuronal and LymphoidCells. Alan R. Liss, Inc., N.Y., 215-236). Expression of themacrophage-specific differentiation marker, Mac-1, was examined byimmunofluorescence following treatment of cells for 2 days with 100 nMTPA (Ball et al, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 5374-5378).

EXAMPLE Construction of Recombinant Plasmids and Riboprobe Synthesis

[0032] The plasmid vector p80, which contains the entire human c-fesgenomic sequence (Trus et al, supra), was s digested with Kpn I and XbaI. The resulting 1175 bp fragment, which contains c-fes exons 2 and 3,was inserted into the polylinker region of pGEM4Z (Promega Biotec,Madison, Wis.) (FIG. 1). This recombinant plasmid was named pcfes4ZKX.To prepare a template for riboprobe synthesis, pcfes4ZKX was digestedwith Bgl II and Xba I, which removed c-fes exon 3 and about two-thirdsof intron 2. The terminal Bgl II and Xba I sites were filled in with theKlenow fragment of DNA polymerase, and the plasmid was re-circularizedwith T4 DNA ligase. The resulting recombinant plasmid, pcfes4ZKB,contains c-fes exon 2 flanked by partial sequences of introns 1 and 2(FIG. 2). The c-fes insert is upstream from and in opposite orientationto the bacteriophage T7 promoter. Prior to riboprobe synthesis,pcfes4ZKB was digested to completion with Eco RI, which cuts the plasmid5′ to the c-fes insert.

[0033] Riboprobe synthesis was conducted in a 20 μl reaction containing40 mm Tris-HCl, pH 7.5, 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl, 10 mMDTT, 40 units RNasin, 0.5 mM ATP, UTP, and GTP, 12 μM CTP, 50 μCi[α−³²P]CTP (800 Ci/mmol), and 1.0 μg linearized template DNA (FIG. 3).Reactions were initiated by adding 20 units of T7 RNA polymerase.incubated at 37° C. for 1 h. and terminated by the addition of 5 unitsof RQ1 DNase (Promega). Following DNase treatment for 15 min at 37° C.,2 μg of carrier tRNA were added, the reaction mixture was extracted withphenol-chloroform, and the labeled RNA was precipitated with ethanol.The riboprobe was re-dissolved in 100 μl water, and the amount oflabeled CTP incorporated was determined by TCA precipitation (typically10⁵ to 10⁹ cpm/μg RNA). The c-fes riboprobe synthesized in this manneris 498 nucleotides in length, as it contains some sequences transcribedfrom the parent vector. Probes were prepared on the same day they wereto be used, and the best results were obtained with fresh isotope. Thisprocedure is a modification of the method originally described by Meltonet al (1984) Nucleic Acids Res. 12, 7035-7056.

[0034] A deposit of the recombinant plasmid pcfes4ZKB has been made atthe ATCC, Rockville, Md., on May 19, 1989, under the accession number40610. The deposit shall be viably maintained, replaced if it becomesnon-viable during the life of the patent, for a period of 30 years fromthe date of the deposit, or for 5 years from the last date of requestfor a sample of the deposit, whichever is longer, and upon issuance ofthe patent made available to the public without restriction inaccordance with the provisions of the law. The Commissioner of Patentsand Trademarks, upon request, shall have access to the deposit.

RESULTS Transfection of Cos-1 cells with pECE/fes

[0035] The 13.2 kb Eco RI fragment identified previously as the humanc-fes gene (Trus et al, supra: Roebroek et al, supra) was cloned intothe SV40-based mammalian expression vector pECE (Ellis et al, supra) anddesignated pECE/fes. To test this construct, Cos-1 cells weretransfected by calcium phosphate precipitation and 48 hr later, TritonX-100 extracts of cellular proteins were analyzed for immunoreactivep93^(c-fes) and for tyrosine kinase activity. Cos-1 cells transfectedwith pECE/fes expressed a 93 kDa protein which was specificallyrecognized on Western blots by the c-fes polyclonal antibody (resultsnot shown). Extracts prepared from Cos-1 cells transfected with pECE/fesexpressed a single species of tyrosine kinase activity that was presentin the 1.0% Triton X-100 cell extract (results not shown). These resultsindicated that Cos-1 cells are capable of expressing the genomic DNAencoding c-fes and transcribing a functional gene product. However,Cos-1 cells did not acquire characteristics of myeloid cells as a resultof c-fes transfection.

Co-transfection of K562 Cells with pECE/fes and DSV-2/neo

[0036] Since K562 cells do not express p93^(c-fes), they are an idealcell line for transfection experiments with pECE/fes. K562 cells wereco-transfected with pECE/fes and pSV-2/neo by protoplast fusion and wereselected by cloning in soft agar containing 2.5 mg/ml G418. After 14days in culture, G418-resistant colonies were selected and amplified inRPMI-1640 medium. One percent Triton X-100 cell extracts representingthe membrane fraction of the cell were prepared from G418-resistantcolonies and were screened for tyrosine kinase activity with thenondenaturing gel assay (FIG. 4). Stably transfected colonies designatedWS-1, WS-5, WS-6, and WD-7 had high levels of tyrosine kinase activity.Colony WS-1 expressed a level of tyrosine kinase activity comparable tothat present in HL-60 cells treated with 1.6% Me₂SO (FIG. 5), atreatment which produces granulocytic differentiation (Zylber-Katz etal, 1985, Cancer Res. 45, 5159-5164). p93^(c-fes) tyrosine kinaseactivity was not present in either parental or pSV-2/neo-transfectedK562 cells (FIG. 5).

[0037] A Southern blot of the DNA prepared from several colonies ofstably transfected K562 cells indicated varying levels of integration ofthe c-fes gene (FIG. 6). The most dramatic example is seen intransfected clone WS-1, in which the level of the c-fes gene is morethan 30 times higher than that of the K562 wild-type cells. Theintensity of the hybridization signal was similar to the level oftyrosine kinase activity expressed by the various clones (FIG. 4).Digestion of WS-1 cell DNA with Eco RI and Xho I generated the expected13.2 kb and 4.4 kb fragments that were identical to those present in p80following hybridization with the v-fes DNA probe (FIG. 7).

Analysis of c-fes transcript levels, mRNA processing, andp93^(c-fes)protein synthesis in K562/fes clones

[0038] Steady-state levels of c-fes mRNA were determined in transfectedK562 clones using the RNase protection assay. The probe used in thisassay is an anti-sense RNA transcript 498 nucleotides in lengthcontaining the 222 nucleotide sequence complimentary to c-fes exon 2.The remainder of the probe Us made up of 5′ and 3′ sequencescomplimentary to c-fes introns 2 and 3, and 37 nucleotides transcribedfrom the vector template. Poly-A⁻ RNA was prepared from K562/fes clonesWS-1, WS-5, and WS-6 and hybridized to the c-fes riboprobe overnight.Following RNase digestion, polyacrylamide/urea gel electrophoresisrevealed a major protected fragment 222 nucleotides in length in each ofthe transfected clones, which corresponds to c-fes exon 2 (FIG. 8). Theintensity of this band is proportional to the level of c-fes genomicintegration (FIG. 6), p93^(c-fes) protein levels (see below). Note thatan identical protected fragment is present following the RNaseprotection assay of poly-A³¹ RNA from HL-60 cells, a cell line whichnormally expresses p93^(c-fes). By contrast, no protected fragments wereobserved following the RNase protection assay of untransfected K562cells.

[0039] In addition to the major band of 222 nucleotides, K562/fes clonesWS-1 and WS-5 also exhibited a protected fragment of 460 nucleotides(FIG. 8), which corresponds to the size of the c-fes genomic fragmentcontained within the probe (i.e., intron and exon sequences). Thisindicates that a significant fraction of the c-fes mRNA from transfectedcells contains intron sequences. and suggests that c-fes mRNA is lessefficiently processed in the transfected clones than in HL-60 cells.which do not exhibit this band. Minor protected fragments approximately320 and 370 nucleotides in length are also visible in transfected clonesWS-1 and WS-5, as well as in HL-60 cells. These fragments may arise fromalternate processing of the primary c-fes transcript that occurs 5′ toexon 2, as several alternate splice acceptor sites have been proposed inintron 2 of the c-fes genomic sequence (Roebroek et al, supra).

[0040] The results indicate that the translation of c-fes ARENA intop93^(c-fes) protein proceeds normally in transfected K562 cells. Thisconclusion is based on immunoprecipitation experiments using ananti-v-fes monoclonal antibody. As shown in FIG. 9, transfected K562clones WS-1 and WS-5 express an immunoreactive 93 kDa protein not seenin the K562 wild type cells. Note that an immunoreactive protein ofidentical electrophoretic mobility is also seen in immunoprecipitates ofHL-60 cells, which are enriched in p93^(c-fes).

Phenotypic alterations in colonies of K562 cells transfected with c-fes

[0041] Clones WS-1, WS-5 and WS-6 were selected for further study of thechanges in maturation which accompanied selection of these cell lines.After 2-3 passages, WS-1 cells grew at a slower rate than wild type K562cells (FIG. 10), a property which may be indicative of differentiation.In addition, all clones adhered loosely to the culture flask, a propertywhich was not seen with parental or pSV-2/neo transfected cells (TableI). Most notable was the response of WS-1 and WS-5 cells to TPA, atreatment which produced approximately 50% macrophage-like cells (FIGS.11A,B). TPA-treatmenrt of transfected K562 cells also resulted inexpression of the macrophage-specific differentiation antigenMac-1(Springer et al, 1979, Eur. J. Immunol. 9, 301-306), whereasTPA-treated wild type cells displayed almost no detectable Mac-1imnunofluorescence (Table I) Several functional parameters which areindicative of mature myeloid cells were also examined.Erythrophagocytosis increased dramatically in clones WS-1 and WS-5 andto a lesser extent in WS-6 (Table I and FIGS. 11E, F). The percentage ofFc receptor positive cells is high in K562 cells (Koeffler et al, 1981,Cancer Res. 41, 919-926) but doubled in all the clones (Table I).Several enzymatic features of mature myeloid cells were also acquired.In parental K562 cells, lysozyme activity was absent, but it was readilydetected in all selected clones, and all transfected cell linesdemonstrated high levels of NBT reduction.

[0042] Although transfected K562 cells express mature myeloidcharacteristics and respond to TPA, they still retain the ability toundergo erythroid differentiation in response to hemin. Treatment ofK562, K562/WS-1, and K562/WS-5 with 100 mM hemin for 5 days resulted in64%, 49% and 63% benzidine-positive cells, respectively.

[0043] In summary, the results presented herein clearly indicate thatthe differentiation-associated 93 kDa tyrosine kinase activity is theproduct of the human c-fes gene. Expression of p93^(c-fes) is found tobe especially high in mature peripheral monocytes and granulocytes,acute and chronic myelogenous leukemias and in leukemia cell linescapable of myeloid differentiation such as K562 and Kg-1a, p93^(c-fes)expression is either very low or absent. These findings suggest thatp93^(c-fes) plays a definitive role during the process of maturation ofmyeloid cells.

[0044] The K562 leukemia cell line provided a convenient model to studythe function of the human c-fes gene and its role in myeloiddifferentiation. This cell line does not express p93^(c-fes) and cannotbe induced to differentiate along the granulocyte/monocyte pathway by avariety of differentiating agents (Koeffler et al, supra). Therefore,this cell line was utilized herein for transfection with the human c-fesgene in order to identify the role of c-fes in the differentiationprocess. It was observed that K562 cells transfected with the c-fes geneexpressed an active p93^(c-fes) tyrosine kinase which coincided with theexpression of phenotypic markers indicative of a more differentiatedcell type such as increased phagocytosis, Fc receptors, NBT reductionand lysozyme activity. . The latter activity in clone WS-1 wascomparable to levels found in mature leukocytes. This clonal cell linealso responded dramatically to the phorbol ester, TPA. resulting in itsmorphologic transformation to a macrophage-like cell and expression ofthe macrophage surface antigen, Mac-1. Thus, these results demonstratethat an active c-fes gene is imperative for the ultimate expression ofthe mature myeloid phenotype.

[0045] For the purpose of routine assays for the detection of c-fesmRNA, non-radioactive riboprobes are easily prepared as follows.

Nonradioactive RNA probe synthesis

[0046] Two procedure can be employed to prepare nonradioactive RNAprobes. The first procedure will utilize a 20 μl reaction containing 40mM Tris-HCl, pH 7.5, 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl, 10 mM DTT,40 units RNasin. 0.5 mM ATP, CTP, and GTP, 0.5 mM5(N-[N-biotinyl-ε-aminocaproyl]-3-aminoallyl)-uridine 5′- triphosphate,and 1.0 μg linearized template DNA. Reactions are initiated by adding 20units of T7 RNA polymerase, incubated at 37° C. for 1 h, and terminatedby the addition of units of RQ1 DNase (Promega). Following DNasetreatment for 15 min at 37° C., 2 μg of carrier tRNA is added, thereaction mixture is extracted with phenol/chloroform, and the labeledRNA is precipitated with ethanol.

[0047] The second method will employ the same reaction mixture exceptthat 0.5 mM UTP Is substituted for5-(N-[N-biotinyl-ε-aminocaproyl]-3-aminoallyl)-uridine 5′-triphosphate.Following precipitation with ethanol as described above, the RNA isreacted with Photoprobe Biotin (Vector Labs, Burlingame, Calif.), aphotoactivatable form of biotin which covalently labels the RNA probe.

[0048] In both instances, the biotinylated RNA probe used in the RNaseprotection assay is detected with a strepavidin-immunoglobulin-alkalinephosphatase conjugate utilizing NBT and BCIP for color detection (Oncor,Gaithersburg, Md.). However, other methods of color detection can, ofcourse, also be employed as will be suggested to one of ordinary skillin the art.

[0049] A kit for the detection of c-fes mRNA comprises a containercontaining the riboprobe of the present invention, either prepared freshor cryopreserved.

[0050] A method for the detection of c-fes mRNA in situ or in vitrocomprises reacting a cell or tissue preparation with the radioactive ornon-radioactive riboprobe of the present invention and determining thedegree of hybridization by standard methodologies well known to one ofordinary skill in the art. Such methodologies include radiolabeled,immuno-histochemical, fluorescence measurement and the like.

[0051] Of course, the present invention now makes it possible to inducemyelopoiesis in immature myeloid cells by introducing genomic c-fes genein immature myeloid cells in which myeloid differentiation is desired.

[0052] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. TABLE I Phenotype ofparental K562 cells and colonies transfected with c-fes DifferentiationMarker^(a) HL-60 K562 K562/neo WS-1 WS-5 WS-6 percent positivePhagocytosis 42^(b) 0 1 65 56 12 Fc receptors 68^(b) 52 48 94 85 81 NBTreduction 77^(b) 1 3 64 43 38 Lysozyme n.d.^(d) 0 0 4.6 3.6 4.5activity^(c) Adherence 0^(b) 0 0 80 80 65 Response to 10⁻⁷ M TPA:Adherence 75 0 0 50 56 n.d. Mac-1 88 2 3 70 73 18

What is claimed is:
 1. A kit for the detection of c-fes mRNA, comprisinga container containing an RNA sequence that hybridizes specifically withhuman c-fes mRNA.
 2. A method of detecting the prescence of c-fes mRNA,comprising hybridizing RNA in a biological sample in which the prescenceof c-fes mRNA is to be ascertained, with an RNA sequence that hybridizesspecifically with human c-fes mRNA and determining the occurrence ofhybridization with said RNA sequence by conventional methodologies, apositive hybridization reaction being indicative of the presence ofc-fes mRNA in said sample.